Methods and apparatus for synthesizing nucleic acid

ABSTRACT

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Non-provisionalpatent application Ser. No. 14/459,014, filed Aug. 13, 2014, which is acontinuation-in-part of U.S. Non-provisional patent application Ser. No.14/056,687, filed Oct. 17, 2013, now issued as U.S. Pat. No. 8,808,989,which claims the benefit of and priority to U.S. Provisional PatentApplication Ser. No. 61/891,162, filed Oct. 15, 2014 and U.S.Provisional Patent Application Ser. No. 61/807,327, filed Apr. 2, 2013.This application additionally claims priority to U.S. Provisional PatentApplication Ser. No. 62/069,067, filed Oct. 27, 2014 and U.S.Provisional Patent Application Ser. No. 62/038,604, filed Aug. 18, 2014.The contents of each of the above applications are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The invention relates to methods and apparatus for synthesizingpolynucleotides (de novo) with a desired sequence and without the needfor a template. As such, the invention provides the capacity to makelibraries of polynucleotides of varying sequence and varying length forresearch, genetic engineering, and gene therapy.

BACKGROUND

Genetic engineering requires tools for determining the content ofgenetic material as well as tools for constructing desired geneticmaterials. The tools for determining the content of genetic materialhave made it possible to sequence an entire human genome in about oneday for under $1,000. (See Life Technologies, Press Release: BenchtopIon Proton™ Sequencer, Jan. 10, 2012). In contrast, the tools forconstructing desired genetic materials, e.g., de novo DNA synthesis,have not improved at the same pace. As a point of reference, over thepast 25 years, the cost (per base) of de novo small nucleic acidsynthesis has dropped 10-fold, while the cost (per base) of nucleic acidsequencing has dropped over 10,000,000-fold. The lack of progress in DNAsynthesis now limits the pace of translational genomics, i.e., wherebythe role of individual sequence variations are determined and used todevelop therapeutic treatments.

Currently, most de novo nucleic acid sequences are synthesized usingsolid phase phosphoramidite-techniques developed more than 30 years ago.The technique involves the sequential de-protection and synthesis ofsequences built from phosphoramidite reagents corresponding to natural(or non-natural) nucleic acid bases. Phosphoramidite nucleic acidsynthesis is length-limited, however, in that nucleic acids greater than200 base pairs (bp) in length experience high rates of breakage and sidereactions. Additionally, phosphoramidite synthesis produces toxicby-products, and the disposal of this waste limits the availability ofnucleic acid synthesizers, and increases the costs of contract oligoproduction. (It is estimated that the annual demand for oligonucleotidesynthesis is responsible for greater than 300,000 gallons of hazardouschemical waste, including acetonitrile, trichloroacetic acid, toluene,tetrahydrofuran, and pyridine. See LeProust et al., Nucleic Acids Res.,vol. 38(8), p. 2522-2540, (2010), incorporated by reference herein inits entirety). Thus, there is a need for more efficient andcost-effective methods for oligonucleotide synthesis.

SUMMARY

The invention provides improved methods for nucleic acid synthesis.Methods of the invention provide faster and longer de novo synthesis ofpolynucleotides. As such, the invention dramatically reduces the overallcost of synthesizing custom nucleic acids. Methods of the invention aredirected to template-independent synthesis of polynucleotides by using anucleotidyl transferase enzyme to incorporate nucleotide analogs havingan unmodified 3′ hydroxyl coupled to an inhibitor by a cleavable linker.Because of the inhibitor, synthesis pauses with the addition of each newbase, whereupon the linker is cleaved, separating the inhibitor andleaving a polynucleotide that is essentially identical to a naturallyoccurring nucleotide (i.e., is recognized by the enzyme as a substratefor further nucleotide incorporation).

The invention additionally includes an apparatus that utilizes methodsof the invention for the production of custom polynucleotides. Anapparatus of the invention includes one or more bioreactors providingaqueous conditions and a plurality of sources of nucleotide analogs. Thebioreactor may be e.g., a reservoir, a flow cell, or a multi-well plate.Starting from a solid support, the polynucleotides are grown in thereactor by adding successive nucleotides via the natural activity of anucleotidyl transferase, e.g., a terminal deoxynucleotidyl transferase(TdT) or any other enzyme which elongates DNA or RNA strands withouttemplate direction. Upon cleavage of the linker, a naturalpolynucleotide is exposed on the solid support. Once the sequence iscomplete, the support is cleaved away, leaving a polynucleotideessentially equivalent to that found in nature. In some embodiments, theapparatus is designed to recycle nucleotide analog solutions byrecovering the solutions after nucleotide addition and reusing solutionsfor subsequence nucleotide addition. Thus, less waste is produced, andthe overall cost per base is reduced as compared to state-of-the-artmethods. In certain embodiments, a bioreactor may include a microfluidicdevice and/or use inkjet printing technology.

Terminating groups may include, for example, charged moieties or stericinhibitors. In general, large macromolecule that prevent nucleotidyltransferase enzymes from achieving a functional conformation can be usedto inhibit oligonucleotide synthesis. Such macromolecules may includepolymers, polypeptides, polypeptoids, and nanoparticles. Themacromolecules should be large enough to physically block access to theactive site of the nucleotidyl transferase, not so large as tonegatively alter the reaction kinetics. The macromolecules can be linkedto nucleotide analogs using a variety of linking molecules, as describedbelow.

In some embodiments, oligonucleotide synthesis may include introductionof a 3′ exonuclease to the one or more synthesized oligonucleotidesafter each nucleotide analog addition, but before cleaving theterminating group. The terminating group may block the 3′ exonucleasefrom acting on the oligonucleotides which have successfully addeduncleaved nucleotide analogs, while oligonucleotides that have notsuccessfully added the inhibitor will be removed by the 3′ exonuclease.In this manner, the invention allows for in-process quality control andmay eliminate the need for post-synthesis purification.

Other aspects of the invention are apparent to the skilled artisan uponconsideration of the following figures and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a genus of deoxycytidine triphosphate (dCTP) analogshaving a cleavable terminator linked at the N-4 position;

FIG. 1B shows cleavage of the cleavable terminator from a dCTP analog ofFIG. 1A to achieve a “natural” dCTP and a cyclic leaving molecule;

FIG. 2A shows a genus of deoxyadenosine triphosphate (dATP) analogshaving a cleavable terminator linked at the N-6 position;

FIG. 2B shows cleavage of the cleavable terminator from a dATP analog ofFIG. 2A to achieve a “natural” dATP and a cyclic leaving molecule;

FIG. 3A shows a genus of deoxyguanosine triphosphate (dGTP) analogshaving a cleavable terminator linked at the N-2 position;

FIG. 3B shows cleavage of the cleavable terminator from a dGTP analog ofFIG. 3A to achieve a “natural” dGTP and a cyclic leaving molecule;

FIG. 4A shows a genus of deoxythymidine triphosphate (dTTP) analogshaving a cleavable terminator linked at the N-3 position;

FIG. 4B shows cleavage of the cleavable terminator from a dTTP analog ofFIG. 4A to achieve a “natural” dTTP and a cyclic leaving molecule;

FIG. 5A shows a genus of deoxyuridine triphosphate (dUTP) analogs havinga cleavable terminator linked at the N-3 position;

FIG. 5B shows cleavage of the cleavable terminator from a dUTP analog ofFIG. 5A to achieve a dUTP and a cyclic leaving molecule;

FIG. 6 shows an exemplary deoxycytidine triphosphate (dCTP) analoghaving a Staudinger linker connecting a blocking Asp-Asp molecule to theN-4 position of the deoxycytidine and subsequent cleavage of theStaudinger linker under aqueous conditions to achieve a dCTP and aleaving group;

FIG. 7A shows a genus of cytidine triphosphate (rCTP) analogs having acleavable terminator linked at the N-4 position;

FIG. 7B shows cleavage of the cleavable terminator from a rCTP analog ofFIG. 7A to achieve a “natural” rCTP and a cyclic leaving molecule;

FIG. 8A shows a genus of adenosine triphosphate (rATP) analogs having acleavable terminator linked at the N-6 position;

FIG. 8B shows cleavage of the cleavable terminator from an rATP analogof FIG. 8A to achieve a “natural” rATP and a cyclic leaving molecule;

FIG. 9A shows n genus of guanosine triphosphate (rGTP) analogs having acleavable terminator linked at the N-2 position;

FIG. 9B shows cleavage of the cleavable terminator from a rGTP analog ofFIG. 9A to achieve a “natural” rGTP and a cyclic leaving molecule;

FIG. 10A shows a genus of thymidine triphosphate (rTTP) analogs having acleavable terminator linked at the N-3 position;

FIG. 10B shows cleavage of the cleavable terminator from a rTTP analogof FIG. 10A to achieve a “natural” rTTP and a cyclic leaving molecule;

FIG. 11A shows a genus of uridine triphosphate (rUTP) analogs having acleavable terminator linked at the N-3 position;

FIG. 11B shows cleavage of the cleavable terminator from a rUTP analogof FIG. 11A to achieve a rUTP and a cyclic leaving molecule;

FIG. 12 shows an exemplary cytidine triphosphate (rCTP) analog having aStaudinger linker connecting a blocking Asp-Asp molecule to the N-4position of the cytidine and subsequent cleavage of the Staudingerlinker under aqueous conditions to achieve a rCTP and a leaving group;

FIG. 13 shows an exemplary terminal deoxynucleotidyl transferase (TdT)mediated polynucleotide synthetic cycle, including: (a) incorporation ofa nucleotide triphosphate analog comprising cleavable terminator,dN*TP—OH, and (b) removal of the terminating blocking group (indicatedby *), thus enabling the next dN*TP—OH to be incorporated, wherein N=A,G, C, or T;

FIG. 14 shows an exemplary nucleotide analog with a cleavable linkercomprising a variable number of methylene bridges;

FIG. 15 shows an exemplary nucleotide analog with a cleavable linkercomprising a cysteine residue;

FIG. 16A shows an exemplary nucleotide analog with an anionic inhibitorcomprising a single phosphate group;

FIG. 16B shows an exemplary nucleotide analog with an anionic inhibitorcomprising two phosphate groups;

FIG. 16C shows an exemplary nucleotide analog with an anionic inhibitorcomprising three phosphate groups;

FIG. 17 shows an exemplary microfluidic polynucleotide synthesis device;

FIG. 18 shows an exemplary polypeptoid inhibitor suitable for use in theinvention;

FIG. 19 shows a flow-chart describing the use of a 3′ exonuclease todigest oligonucleotides that are not properly terminated betweenoligonucleotide synthesis cycles.

DETAILED DESCRIPTION

The invention provides improved methods for synthesizingpolynucleotides, such as DNA and RNA, using enzymes and nucleic acidanalogs. Using the disclosed methods, specific sequences ofpolynucleotides can be synthesized de novo, base by base, in an aqueousenvironment, without the use of a nucleic acid template. Additionally,because the nucleotide analogs have an unmodified 3′ hydroxyls, i.e., asfound in “natural” deoxyribose and ribose molecules, the analogs resultin “natural” nucleotides when an inhibitor, also referred to herein as aterminator or terminator group attached via a cleavable linker, isremoved from the base. Other nucleotide analogs can also be used which,for example, include self-eliminating linkers, or nucleotides withmodified phosphate groups. In most instances, the blocking group isdesigned to not leave behind substantial additional molecules, i.e.,designed to leave behind “scarless” nucleotides that are recognized as“natural” nucleotides by the enzyme. Thus, at the conclusion of thesynthesis, upon removal of the last blocking group, the synthesizedpolynucleotide is chemically and structurally equivalent to thenaturally-occurring polynucleotide with the same sequence. The syntheticpolynucleotide can, thus, be incorporated into living systems withoutconcern that the synthesized polynucleotide will interfere withbiochemical pathways or metabolism.

The process and analogs of the current invention can be used for thenon-templated enzymatic synthesis of useful oligo- andoligodeoxynucleotides especially of long oligonucleotides (<5000 nt).Products can be single strand or partially double strand depending uponthe initiator used. The synthesis of long oligonucleotides requires highefficiency incorporation and high efficiency of reversible terminatorremoval. The initiator bound to the solid support consists of a short,single strand DNA sequence that is either a short piece of the userdefined sequence or a universal initiator from which the user definedsingle strand product is removed.

In one aspect, the disclosed methods employ commercially-availablenucleotidyl transferase enzymes, such as terminal deoxynucleotidyltransferase (TdT), to synthesize polynucleotides from nucleotide analogsin a step-by-step fashion. The nucleotide analogs are of the form:NTP-linker-inhibitorwherein NTP is a nucleotide triphosphate (i.e., a dNTP or an rNTP), thelinker is a cleavable linker between the pyridine or pyrimidine of thebase, and the inhibitor is a group that prevents the enzyme fromincorporating subsequent nucleotides. At each step, a new nucleotideanalog is incorporated into the growing polynucleotide chain, whereuponthe enzyme is blocked from adding an additional nucleotide by theinhibitor group. Once the enzyme has stopped, the excess nucleotideanalogs can be removed from the growing chain, the inhibitor can becleaved from the NTP, and new nucleotide analogs can be introduced inorder to add the next nucleotide to the chain. By repeating the stepssequentially, it is possible to quickly construct nucleotide sequencesof a desired length and sequence. Advantages of using nucleotidyltransferases for polynucleotide synthesis include: 1) 3′-extensionactivity using single strand (ss) initiating primers in atemplate-independent polymerization, 2) the ability to extend primers ina highly efficient manner resulting in the addition of thousands ofnucleotides, and 3) the acceptance of a wide variety of modified andsubstituted NTPs as efficient substrates. In addition, the invention canmake use of an initiator sequence that is a substrate for nucleotidyltransferase. The initiator is attached to a solid support and serves asa binding site for the enzyme. The initiator is preferably a universalinitiator for the enzyme, such as a homopolymer sequence and isrecyclable on the solid support, the formed oligonucleotide beingcleavable from the initiator.

Methods of the invention are well-suited to a variety of applicationsthat currently use synthetic nucleic acids, e.g.,phosphoramidite-synthesized DNA oligos. For example, polynucleotidessynthesized with the methods of the invention can be used as primers fornucleic acid amplification, hybridization probes for detection ofspecific markers, and for incorporation into plasmids for geneticengineering. However, because the disclosed methods produce longersynthetic strings of nucleotides, at a faster rate, and in an aqueousenvironment, the disclosed methods also lend themselves tohigh-throughput applications, such as screening for expression ofgenetic variation in cellular assays, as well as synthetic biology.Furthermore, the methods of the invention will provide the functionalityneeded for next-generation applications, such as using DNA as syntheticread/write memory, or creating macroscopic materials synthesizedcompletely (or partially) from DNA.

The invention and systems described herein provide for synthesis ofpolynucleotides, including deoxyribonucleic acids (DNA) and ribonucleicacids (RNA). While synthetic pathways for “natural” nucleotides, such asDNA and RNA, are described in the context of the common nucleic acidbases, e.g., adenine (A), guanine (G), cytosine (C), thymine (T), anduracil (U), it is to be understood that the methods of the invention canbe applied to so-called “non-natural” nucleotides, including nucleotidesincorporating universal bases such as 3-nitropyrrole 2′-deoxynucleosideand 5-nitroindole 2′-deoxynucleoside, alpha phosphorothiolate,phosphorothioate nucleotide triphosphates, or purine or pyrimidineconjugates that have other desirable properties, such as fluorescence.Other examples of purine and pyrimidine bases includepyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g.,8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine,7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. In some instances, itmay be useful to produce nucleotide sequences having unreactive, butapproximately equivalent bases, i.e., bases that do not react with otherproteins, i.e., transcriptases, thus allowing the influence of sequenceinformation to be decoupled from the structural effects of the bases.

Analogs

The invention provides nucleotide analogs having the formulaNTP-linker-inhibitor for synthesis of polynucleotides in an aqueousenvironment. With respect to the analogs of the formNTP-linker-inhibitor, NTP can be any nucleotide triphosphate, such asadenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidinetriphosphate (CTP), thymidine triphosphate (TTP), uridine triphosphate(UTP), nucleotide triphosphates, deoxyadenosine triphosphate (dATP),deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP).

The linker can be any molecular moiety that links the inhibitor to theNTP and can be cleaved. For example, the linkers can be cleaved byadjusting the pH of the surrounding environment. The linkers may also becleaved by an enzyme that is activated at a given temperature, butinactivated at another temperature. In some embodiments, the linkersinclude disulfide bonds.

Linkers may, for example, include photocleavable, nucleophilic, orelectrophilic cleavage sites. Photocleavable linkers, wherein cleavageis activated by a particular wavelength of light, may include benzoin,nitroveratryl, phenacyl, pivaloyl, sisyl, 2-hydroxy-cinnamyl,coumarin-4-yl-methyl, or 2-nitrobenzyl based linkers.

Examples of nucleophilic cleavage sites include fluoride ion cleavablesilicon-oxygen bonds or esters which may be cleaved in a basic solution.Electrophilically cleaved linkers may include acid induced cleavagesites which may comprise trityl, tert-butyloxycarbonyl groups, acetalgroups, and p-alkoxybenzyl esters and amides. In certain aspects, acleavable linker may include a cysteine residue as shown in FIG. 15.

The linker can be attached, for example, at the N4 of cytosine, the N3or O4 of thymine, the N2 or N3 of guanine, and the N6 of adenine, or theN3 or O4 of uracil because attachment at a carbon results in thepresence of a residual scar after removal of the polymerase-inhibitinggroup. The linker is typically on the order of at least about 10Angstroms long, e.g., at least about 20 Angstroms long, e.g., at leastabout 25 Angstroms long, thus allowing the inhibitor to be far enoughfrom the pyridine or pyrimidine to allow the enzyme to bind the NTP tothe polynucleotide chain via the attached sugar backbone. In someembodiments, the cleavable linkers are self-cyclizing in that they forma ring molecule that is particularly non-reactive toward the growingnucleotide chain.

In certain aspects, a cleavable linker may include a variable number ofmethylene bridges on the NTP or the inhibitor side of a disulfide bond,including, for example, 1, 2, 3, or 4 methylene bridges as shown inFIGS. 14 and 16A-C. These methylene bridges may be used to increase thespace between the NTP and the inhibitor. As noted above, the length ofthe cleavable linker may be selected in order to prevent the inhibitorfrom interfering with coupling of the NTP to the synthesizedpolynucleotide. In some embodiments of the invention, the distance ofthe charged group to the NTP plays an important role in theeffectiveness of inhibiting a subsequent nucleotide incorporation.

For example, in some embodiments using a charged moiety as an inhibitor,the charged moiety may be from about 5 to about 60 bonds away from theNTP. In some other embodiments, the charged moiety of the inhibitor maybe from about 10 to about 40 bonds away from the NTP. In some otherembodiments, the charged moiety of the inhibitor can be from about 10 toabout 35 bonds away from the NTP. In some other embodiments, the chargedmoiety of the inhibitor may be from about 10 to about 30 bonds away fromthe NTP. In some other embodiments, the charged moiety of the inhibitoris from about 10 to about 20 bonds away from the NTP. The number ofbonds between the charged moiety and the NTP may be increased byincluding additional methylene bridges.

The nucleotide analogs can include any moiety linked to the NTP thatinhibits the coupling of subsequent nucleotides by the enzyme. Theinhibitory group can be a charged group, such as a charged amino acid,or the inhibitory group can be a group that becomes charged dependingupon the ambient conditions. In some embodiments, the inhibitor mayinclude a moiety that is negatively charged or capable of becoming anegatively charged. For example, an inhibitor may include a chain ofphosphate groups (e.g., 1, 2, or 3, phosphates) as shown in FIGS. 16A-C,wherein additional phosphates increase the overall anionic charge of theinhibitor. In other embodiments, the inhibitor group is positivelycharged or capable of becoming positively charged. In some otherembodiments, the inhibitor is an amino acid or an amino acid analog. Theinhibitor may be a peptide of 2 to 20 units of amino acids or analogs, apeptide of 2 to 10 units of amino acids or analogs, a peptide of 3 to 7units of amino acids or analogs, a peptide of 3 to 5 units of aminoacids or analogs. In some embodiments, the inhibitor includes a groupselected from the group consisting of Glu, Asp, Arg, His, and Lys, and acombination thereof (e.g., Arg, Arg-Arg, Asp, Asp-Asp, Asp, Glu,Glu-Glu, Asp-Glu-Asp, Asp-Asp-Glu or AspAspAspAsp, etc.). Peptides orgroups may be combinations of the same or different amino acids oranalogs. In certain embodiments, a peptide inhibitor may be acetylatedto discourage errant bonding of free amino groups. The inhibitory groupmay also include a group that reacts with residues in the active site ofthe enzyme thus interfering with the coupling of subsequent nucleotidesby the enzyme. The inhibitor may have a charged group selected from thegroup consisting of —COO, —NO₂, —PO₄, —PO₃, —SO₂, or —NR₃ where each Rmay be H or an alkyl group. In other embodiments, the inhibitor moietydoes not comprise a —PO4 group.

In certain aspects, a terminator or inhibitor may include a stericinhibitor group. Such a steric inhibitor group may allow for theNTP-linker-inhibitor (i.e., nucleotide analog) to be incorporated ontothe unblocked 3′ OH of an oligonucleotide, said incorporation beingcatalyzed by nucleotidyl transferase. The steric inhibitor group mayphysically block the incorporation of nucleotides or additionalnucleotide analogs onto the unblocked 3′ OH of the incorporatednucleotide analog. Steric inhibitors may also block 3′ endonucleasesfrom acting on a nucleotide analog and, accordingly, on oligonucleotidesto which an un-cleaved nucleotide analog has been incorporated.

Steric inhibitors can include, for example, chemical polymers,nanoparticles, poly-N-substituted glycines (peptoids), or proteins. Asteric inhibitor of the invention may be a variety of sizes including,e.g., greater than 20 Å, greater than 30 Å, greater than 40 Å, greaterthan 50 Å, greater than 60 Å, greater than 70 Å, greater than 80 Å,greater than 90 Å, greater than 100 Å, greater than 110 Å, greater than120 Å, greater than 130 Å, greater than 140 Å, or greater than 150 Å. Inpreferred embodiments, a steric inhibitor may be monodisperse orsubstantially monodisperse. Steric inhibitors may be water soluble andconformationally constrained (i.e., of a rigid or semi-rigid form). Incertain aspects, a steric inhibitor will physically block access to theactive site of the relevant nucleotidyl transferase enzyme because ofthe size or the conformation of the inhibitor. In preferred embodiments,the steric inhibitor may comprise a non-natural bio-inspired polymersuch as a polypeptoid or a non-natural polypeptide.

In certain aspects, a self-assembling polypeptoid sequence may be usedas a steric inhibitor. Peptoid monomers are often based on anN-substituted glycine backbone. Because the backbone is devoid ofhydrogen bond donors, polypeptoids are readily processed while stillbeing able to form secondary structures such as helices. They alsoprovide the beneficial properties of allowing polarities and side chainssimilar to peptides, while being generally chemically and thermallystable. Self-assembling polypeptoid steric inhibitors according to theinvention may self-assemble single peptoid helices to form microspheresin the micrometer range of diameters including, 0.3 μm, 0.4 μm, 0.5 μm,0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, or3.5 μm, among others. In certain aspects, steric inhibitors may includepeptoids with C-α-branched side chains, N-Aryl side chains,N-1-naphthylethyl side chains, or other formations capable of formingstable helical structures. An example of a peptoid steric inhibitor isshown in FIG. 18. FIG. 18 illustrates a branched poly-N-methoxyethylglycine which may be used as a steric inhibitor according to theinvention. In certain embodiments, a steric inhibitor may include areactive group that is easily joined to a linker group, e.g., acleavable linking group as described herein.

In other embodiments, a steric inhibitor may comprise a polymer, such asa biocompatible polymer. The polymer may comprise blocks of differentpolymers, such that the blocks form a desired macroscopic structure,e.g., a sphere when exposed to an aqueous environment. For example, thecopolymer may comprise blocks of hydrophilic and hydrophobic blocks sothat the polymer self-assembles into a micellar structure upon additionto water. In some embodiments, the hydrophobic blocks may be selectedfrom polycaprolactones (PCL), polydimethylsiloxanes (PDMS),polymethylmethacrylate (PMMA), or polylactides (PLA). The hydrophilicblocks may include polyethylene glycol (PEG) or other polyalcohol.

In other embodiments, the inhibitor may comprise a nanoparticle ofsufficient size to block the activity of a nucleotidyl transferase. Suchnanoparticles may comprise, e.g., gold, silver, silicon, cerium oxide,iron oxide, titanium dioxide, silicon nitride, silicon boride, orsilica, e.g., mesoporous silica. In other embodiments, the nanoparticlesmay comprise highly-ordered molecular structures, such as fullerenes,e.g., buckyballs and nanotubes, comprising carbon, or semiconductors.

Steric inhibitors may have no charge or may be positively or negativelycharged to provide compatibility with the nucleotide to which it islinked and with the nucleotidyl transferase enzyme so that the inhibitordoes not interfere with the incorporation reaction on the 5′ end of theNTP analog. Steric inhibitors may incorporate a variety of amino acidresidues in order to provide a desired conformation, charge, orattachment site.

An example of a nucleotide analog of the type NTP-linker-inhibitor isshown in FIG. 1A. The analog in FIG. 1A includes an inhibitory(-Asp-Asp-) group linked to the N4 position of dCTP through a disulfide(—S—S—) bond while providing an unblocked, unmodified 3′-OH on the sugarring. The linker is constructed such that all linker atoms (includingthe 2nd incorporation-inhibiting moiety) can be removed, therebyallowing the nascent DNA strand to revert to natural nucleotides. Asshown in FIG. 1B, an aqueous reducing agent, such astris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), can beused to cleave the —S—S— bond, resulting in the loss of the inhibitorfunction (deblocking). As shown in FIG. 1B, a self-cyclizing linker canbe incorporated, resulting in a cyclic oxidized tetrahydrothiopheneleaving group that is easily removed from the reagent solution at theconclusion of nucleotide synthesis.

Exemplary schemes for synthesizing dCTP analogs of FIG. 1A are shownbelow in Schemes 1A and 1B:

In a fashion analogous to Schemes 1A and 1B, nucleotide analogs of thetype NTP-linker-inhibitor can also be formed by attaching thelinker-inhibitor moiety to the N6 of adenine (FIG. 2), the N2 of guanine(FIG. 3), the N3 of thymine (FIG. 4), or the N3 of uracil (FIG. 5),thereby providing analogs of the “naturally-occurring” dNTPs, as well asa deoxyuracil nucleotide (dUTP). While it is unlikely that there will bewide use of a dUTP, the synthesis is straightforward based upon thechemistry.

The invention is not limited to the linking chemistry of Schemes 1A and1B, however, as carbamate, amide, or other self-eliminating linkagescould also be employed. For example, nucleotides can also be preparedwith Staudinger linkers, as shown in Scheme 2.

A deoxycytidine triphosphate (dCTP) analog created with a Staudingerlinker (Scheme 2) to an Asp-Asp blocking group is shown in FIG. 6. Asshown in FIG. 6, the Staudinger dCTP analog undergoes cleavage underaqueous conditions with the addition of azide and triphenylphosphine.The Staudinger analog shown in FIG. 6 is also suitable for nucleotideextension using nucleotidyl transferases, such as TdT, as describedabove and exemplified in FIGS. 1-5. While not shown explicitly in theFIGS., one of skill in the art can use Scheme 2 in conjunction with thesuitable reactant to produce other nucleotide analogs having Staudingerlinkers as needed for complete de novo nucleotide synthesis. In afashion analogous to FIG. 6, nucleotide analogs of Scheme 2 can beformed by attaching the Staudinger moiety to the N6 of adenine, the N2of guanine, the N3 of thymine, or the N3 of uracil, thereby providinganalogs of the “naturally-occurring” dNTPs, as well as a deoxyuracilnucleotide (dUTP).

The methodologies of Scheme 1A can be used to produce correspondingribonucleotide analogs, e.g., as shown in FIGS. 7-10, by starting withthe appropriate ribonucleotide reactants. Ribonucleotide analogscomprising the Staudinger linker can also be created using Scheme 2 inorder to form the needed ribonucleotide analogs, including, e.g., CTPanalogs, as shown in FIG. 12. Furthermore, all of the ribonucleotideanalogs, i.e., C, A, T, G, U, can be formed using a reaction similar toScheme 2.

Enzymes

The methods of the invention employ nucleotidyl transferases to assemblethe nucleotide analogs into polynucleotides. Nucleotidyl transferasesinclude several families of related transferase and polymerase enzymes.Some nucleotidyl transferases polymerize deoxyribonucleotides moreefficiently than ribonucleotides, some nucleotidyl transferasespolymerize ribonucleotides more efficiently than deoxyribonucleotides,and some nucleotidyl transferases polymerize ribonucleotides anddeoxyribonucleotides at approximately the same rate.

Of particular import to the invention, transferases having polymeraseactivity, such as terminal deoxynucleotidyl transferase (TdT), arecapable of catalyzing the addition of deoxyribonucleotides to the 3′ endof a nucleotide chain, thereby increasing chain length in DNAnucleotides. TdT will only catalyze the addition of 1-2 ribonucleotidesto the growing end of a DNA strand which could be useful in theconstruction of site specific DNA-RNA chimeric polynucleotides. Inparticular, calf thymus TdT, sourced from engineered E. coli, issuitable for use with the invention and available from commercialsources such as Thermo Scientific (Pittsburgh, Pa.). The amino acidsequence corresponding to calf TdT is listed in Table 1 as SEQ ID NO. 1.

TABLE 1  Amino Acid Sequence of Bovine TdTSEQ ID NO. 1: MAQQRQHQRL PMDPLCTASS GPRKKRPRQVGASMASPPHD IKFQNLVLFI LEKKMGTTRR NFLMELARRK GFRVENELSD SVTHIVAENN SGSEVLEWLQ VQNIRASSQL ELLDVSWLIE SMGAGKPVEI TGKHQLVVRT DYSATPNPGF QKTPPLAVKK ISQYACQRKT TLNNYNHIFT DAFEILAENSEFKENEVSYV TFMRAASVLK SLPFTIISMK DTEGIPCLGD KVKCIIEEII EDGESSEVKA VLNDERYQSF KLFTSVFGVG LKTSEKWFRM GFRSLSKIMS DKTLKFTKMQ KAGFLYYEDL VSCVTRAEAE AVGVLVKEAV WAFLPDAFVT MTGGFRRGKKIGHDVDFLIT SPGSAEDEEQ LLPKVINLWE KKGLLLYYDL VESTFEKFKL PSRQVDTLDH FQKCFLILKL HHQRVDSSKS NQQEGKTWKA IRVDLVMCPY ENRAFALLGW TGSRQFERDI RRYATHERKM MLDNHALYDK TKRVFLKAES EEEIFAHLGL  DYIEPWERNAThe nucleotide sequence corresponding to calf TdT is listed in Table 2as SEQ ID NO. 2.

TABLE 2  Nucleic Acid Sequence of Bovine TdTSEQ ID NO. 2: ctcttctgga gataccactt gatggcacagcagaggcagc atcagcgtct tcccatggat ccgctgtgca cagcctcctc aggccctcgg aagaagagac ccaggcaggt gggtgcctca atggcctccc ctcctcatga catcaagttt caaaatttgg tcctcttcat tttggagaag aaaatgggaa ccacccgcag aaacttcctc atggagctgg ctcgaaggaaaggtttcagg gttgaaaatg agctcagtga ttctgtcacc cacattgtag cagaaaacaa ctctggttca gaggttctcg agtggcttca ggtacagaac ataagagcca gctcgcagct agaactcctt gatgtctcct ggctgatcga aagtatgggagcaggaaaac cagtggagat tacaggaaaa caccagcttg ttgtgagaac agactattca gctaccccaa acccaggcttccagaagact ccaccacttg ctgtaaaaaa gatctcccag tacgcgtgtc aaagaaaaac cactttgaac aactataaccacatattcac ggatgccttt gagatactgg ctgaaaattc tgagtttaaa gaaaatgaag tctcttatgt gacatttatg agagcagctt ctgtacttaa atctctgcca ttcacaatca tcagtatgaa ggatacagaa ggaattccct gcctggggga caaggtgaag tgtatcatag aggaaattat tgaagatgga gaaagttctg aagttaaagc tgtgttaaat gatgaacgat atcagtcctt caaactcttt acttctgttt ttggagtggg actgaagaca tctgagaaat ggttcaggat ggggttcaga tctctgagta aaataatgtc agacaaaacc ctgaaattca caaaaatgca gaaagcagga tttctctatt atgaagacct tgtcagctgc gtgaccaggg ccgaagcaga ggcggttggc gtgctggtta aagaggctgt gtgggcattt ctgccggatg cctttgtcac catgacagga ggattccgca ggggtaagaa gattgggcat gatgtagatt ttttaattac cagcccagga tcagcagagg atgaagagca acttttgcct aaagtgataa acttatggga aaaaaaggga ttacttttat attatgacct tgtggagtca acatttgaaa agttcaagtt gccaagcagg caggtggata ctttagatca ttttcaaaaa tgctttctga ttttaaaatt gcaccatcag agagtagaca gtagcaagtccaaccagcag gaaggaaaga cctggaaggc catccgtgtg gacctggtta tgtgccccta cgagaaccgt gcctttgccc tgctaggctg gactggctcc cggcagtttg agagagacat ccggcgctat gccacacacg agcggaagat gatgctggat aaccacgctt tatatgacaa gaccaagagg gtatttctca aagcggaaag tgaagaagaa atctttgcac atctgggatt ggactacatt gaaccatggg aaagaaatgc ttaggagaaa gctgtcaact tttttctttt ctgttctttt tttcaggtta gacaaattat gcttcatatt ataatgaaag atgccttagt caagtttggg attctttaca ttttaccaag atgtagattg cttctagaaa taagtagttt tggaaacgtg atcaggcacc ccctgggtta tgctctggca agccatttgc aggactgatg tgtagaactc gcaatgcatt ttccatagaa acagtgttgg aattggtggc tcatttccag ggaagttcat caaagcccac tttgcccaca gtgtagctga aatactgtat acttgccaat  aaaaatagga aac

While commercially-available TdT is suitable for use with the methods ofthe invention, modified TdT, e.g., having an amino acid sequence atleast 95% in common with SEQ ID NO. 1, e.g., having an amino acidsequence at least 98% in common with SEQ ID NO. 1, e.g., having an aminoacid sequence at least 99% in common with SEQ ID NO. 1, may be used withthe methods of the invention. An organism that expresses a suitablenucleotidyl transferase may comprise a nucleic acid sequence at least95% in common with SEQ ID NO. 2, e.g., at least 98% in common with SEQID NO. 2, e.g., at least 99% in common with SEQ ID NO. 2. In someinstances, a modified TdT will result in more efficient generation ofpolynucleotides, or allow better control of chain length. Othermodifications to the TdT may change the release characteristics of theenzyme, thereby reducing the need for aqueous reducing agents such asTCEP or DTT.

For the synthesis of RNA polynucleotides, a nucleotidyl transferase likeE. coli poly(A) polymerase can be used to catalyze the addition ofribonucleotides to the 3′ end of a ribonucleotide initiator. In otherembodiments, E. coli poly(U) polymerase may be more suitable for usewith the methods of the invention. Both E. coli poly(A) polymerase andE. coli poly(U) polymerase are available from New England Biolabs(Ipswich, Mass.). These enzymes may be used with 3′unblocked reversibleterminator ribonucleotide triphosphates (rNTPs) to synthesize RNA. Incertain embodiments, RNA may be synthesized using 3′blocked, 2′blocked,or 2′-3′blocked rNTPs and poly(U) polymerase or poly(A) polymerase. Theamino acid and nucleotide sequences for E. coli Poly(A) polymerase andE. coli Poly(U) polymerase are reproduced below. Modified E. coliPoly(A) polymerase or E. coli Poly(U) polymerase may be suitable for usewith the methods of the invention. For example, an enzyme, having anamino acid sequence at least 95% in common with SEQ ID NO. 3, e.g.,having an amino acid sequence at least 98% in common with SEQ ID NO. 3,e.g., having an amino acid sequence at least 99% in common with SEQ IDNO. 3, may be used with the methods of the invention. An organism thatexpresses a suitable enzyme may comprise a nucleic acid sequence atleast 95% in common with SEQ ID NO. 4, e.g., at least 98% in common withSEQ ID NO. 4, e.g., at least 99% in common with SEQ ID NO. 4.Alternatively, an enzyme having an amino acid sequence at least 95% incommon with SEQ ID NO. 5, e.g., having an amino acid sequence at least98% in common with SEQ ID NO. 5, e.g., having an amino acid sequence atleast 99% in common with SEQ ID NO. 5, may be used with the methods ofthe invention. An organism that expresses a suitable enzyme may comprisea nucleic acid sequence at least 95% in common with SEQ ID NO. 6, e.g.,at least 98% in common with SEQ ID NO. 6, e.g., at least 99% in commonwith SEQ ID NO. 6.

TABLE 3  Amino Acid Sequence of E. coli Poly(A) polymeraseSEQ ID NO. 3: MFTRVANFCR KVLSREESEA EQAVARPQVTVIPREQHAIS RKDISENALK VMYRLNKAGY EAWLVGGGVR DLLLGKKPKD FDVTTNATPE QVRKLFRNCR LVGRRFRLAH VMFGPEIIEV ATFRGHHEGN VSDRTTSQRG QNGMLLRDNI FGSIEEDAQR RDFTINSLYY SVADFTVRDY VGGMKDLKDG VIRLIGNPET RYREDPVRML RAVRFAAKLG MRISPETAEP IPRLATLLND IPPARLFEES LKLLQAGYGY ETYKLLCEYH LFQPLFPTIT RYFTENGDSP MERIIEQVLK NTDTRIHNDM RVNPAFLFAA MFWYPLLETA QKIAQESGLT YHDAFALAMN DVLDEACRSL AIPKRLTTLT RDIWQLQLRM SRRQGKRAWK LLEHPKFRAA YDLLALRAEV ERNAELQRLV KWWGEFQVSA PPDQKGMLNE LDEEPSPRRR TRRPRKRAPR REGTAThe nucleotide sequence corresponding to E. coli poly(A) polymerase islisted in Table 4 as SEQ ID NO. 4.

TABLE 4  Nucleotide Sequence of E. coli Poly(A) polymeraseSEQ ID NO. 4: atttttaccc gagtcgctaa tttttgccgcaaggtgctaa gccgcgagga aagcgaggct gaacaggcag tcgcccgtcc acaggtgacg gtgatcccgc gtgagcagca tgctatttcc cgcaaagata tcagtgaaaa tgccctgaag gtaatgtaca ggctcaataa agcgggatac gaagcctggc tggttggcgg cggcgtgcgc gacctgttac ttggcaaaaagccgaaagat tttgacgtaa ccactaacgc cacgcctgag caggtgcgca aactgttccg taactgccgc ctggtgggtc gccgtttccg tctggctcat gtaatgtttg gcccggagat tatcgaagtt gcgaccttcc gtggacacca cgaaggtaac gtcagcgacc gcacgacctc ccaacgcggg caaaacggca tgttgctgcg cgacaacatt ttcggctcca tcgaagaaga cgcccagcgc cgcgatttca ctatcaacag cctgtattac agcgtagcgg attttaccgt ccgtgattac gttggcggcatgaaggatct gaaggacggc gttatccgtc tgattggtaa cccggaaacg cgctaccgtg aagatccggt acgtatgctg cgcgcggtac gttttgccgc caaattgggt atgcgcatca gcccggaaac cgcagaaccg atccctcgcc tcgctaccct gctgaacgat atcccaccgg cacgcctgtt tgaagaatcg cttaaactgc tacaagcggg ctacggttac gaaacctata agctgttgtg tgaatatcat ctgttccagc cgctgttccc gaccattacc cgctacttca cggaaaatgg cgacagcccg atggagcgga tcattgaaca ggtgctgaag aataccgata cgcgtatcca taacgatatg cgcgtgaacc cggcgttcct gtttgccgcc atgttctggt acccactgct ggagacggca cagaagatcg cccaggaaag cggcctgacc tatcacgacg ctttcgcgct ggcgatgaac gacgtgctgg acgaagcctg ccgttcactg gcaatcccga aacgtctgac gacattaacc cgcgatatct ggcagttgca gttgcgtatg tcccgtcgtc agggtaaacg cgcatggaaa ctgctggagc atcctaagtt ccgtgcggct tatgacctgt tggccttgcg agctgaagtt gagcgtaacg ctgaactgca gcgtctggtg aaatggtggg gtgagttcca ggtttccgcg ccaccagacc aaaaagggat gctcaacgag ctggatgaag aaccgtcacc gcgtcgtcgt actcgtcgtc cacgcaaacg cgcaccacgt cgtgagggta  ccgcatga

TABLE 5  Amino Acid Sequence of E. coli Poly(U) polymeraseSEQ ID NO. 5: GSHMSYQKVP NSHKEFTKFC YEVYNEIKISDKEFKEKRAA LDTLRLCLKR ISPDAELVAF GSLESGLALK NSDMDLCVLM DSRVQSDTIA LQFYEELIAE GFEGKFLQRA RIPIIKLTSD TKNGFGASFQ CDIGFNNRLA IHNTLLLSSY TKLDARLKPM VLLVKHWAKR KQINSPYFGT LSSYGYVLMV LYYLIHVIKP PVFPNLLLSP LKQEKIVDGF DVGFDDKLED IPPSQNYSSL GSLLHGFFRF YAYKFEPREK VVTFRRPDGY LTKQEKGWTS ATEHTGSADQ IIKDRYILAI EDPFEISHNV GRTVSSSGLY RIRGEFMAAS RLLNSRSYPI PYDSLFEEAThe nucleotide sequence corresponding to E. coli poly(U) polymerase islisted in Table 6 as SEQ ID NO. 6.

TABLE 6  Nucleotide Sequence of E. coli Poly(A) polymeraseSEQ ID NO. 6: ggcagccata tgagctatca gaaagtgccgaacagccata aagaatttac caaattttgc tatgaagtgt ataacgaaat taaaattagc gataaagaat ttaaagaaaa acgcgcggcg ctggataccc tgcgcctgtg cctgaaacgc attagcccgg atgcggaact ggtggcgttt ggcagcctgg aaagcggcct ggcgctgaaa aacagcgata tggatctgtg cgtgctgatg gatagccgcg tgcagagcga taccattgcg ctgcagtttt atgaagaact gattgcggaa ggctttgaag gcaaatttct gcagcgcgcg cgcattccga ttattaaact gaccagcgat accaaaaacg gctttggcgc gagctttcag tgcgatattg gctttaacaa ccgcctggcg attcataaca ccctgctgct gagcagctat accaaactgg atgcgcgcct gaaaccgatg gtgctgctgg tgaaacattg ggcgaaacgc aaacagatta acagcccgta ttttggcacc ctgagcagct atggctatgt gctgatggtg ctgtattatc tgattcatgt gattaaaccg ccggtgtttc cgaacctgct gctgagcccg ctgaaacagg aaaaaattgt ggatggcttt gatgtgggct ttgatgataa actggaagat attccgccga gccagaacta tagcagcctg ggcagcctgc tgcatggctt ttttcgcttt tatgcgtata aatttgaacc gcgcgaaaaa gtggtgacct ttcgccgccc ggatggctat ctgaccaaac aggaaaaagg ctggaccagc gcgaccgaac ataccggcag cgcggatcag attattaaag atcgctatat tctggcgatt gaagatccgt ttgaaattag ccataacgtg ggccgcaccg tgagcagcag cggcctgtat cgcattcgcg gcgaatttat ggcggcgagc cgcctgctga acagccgcag ctatccgatt ccgtatgata  gcctgtttga agaagcg

As discussed above, the inhibitor coupled to the nucleotide analog willcause the transferase, e.g., TdT, to not release from the polynucleotideor prevent other analogs from being incorporated into the growing chain.A charged moiety results in better inhibition, however, researchsuggests that the specific chemical nature of the inhibitor is notparticularly important. For example, both phosphates and acidic peptidescan be used to inhibit enzymatic activity. See, e.g., Bowers et al.,Nature Methods, vol. 6, (2009) p. 593-95, and U.S. Pat. No. 8,071,755,both of which are incorporated herein by reference in their entireties.In some embodiments, the inhibitor will include single amino acids ordipeptides, like -(Asp)₂, however the size and charge on the moiety canbe adjusted, as needed, based upon experimentally determined rates offirst nucleotide incorporation and second nucleotide incorporation. Thatis, other embodiments may use more or different charged amino acids orother biocompatible charged molecule.

Other methods of nucleotide synthesis may be used to build de novooligonucleotides in a template independent fashion using nucleotidyltransferases or modified nucleotidyl transferases. In one embodiment,the polymerase/transferase enzymes can be modified so that they ceasenucleotide addition when they encounter a modification to the phosphateof a 3′-unmodified dNTP analog. This scheme would require a deblockingreagent/reaction that modifies the phosphate end of the nucleotideanalog, which frees up the nascent strand for subsequent nucleotideincorporation. Preferred embodiments of this approach would usenucleotide analogs modified only at the phosphates (alpha, beta orgamma) although modifications of the purine/pyrimidine base of thenucleotide are allowed.

In some embodiments, it may be advantageous to use a 3′ exonuclease toremove oligonucleotides that have not been properly terminated with aninhibitor prior to subsequent nucleotide analog addition. In particular,the inhibitor of the nucleotide analog can be chosen to inhibit theactivity of nucleotidyl transferase and 3′ exonucleases, such that onlyproperly terminated oligonucleotides would be built up. Using thisquality control technique, the purity of the resulting oligonucleotidesequences would be improved. In some embodiments, use of such qualitycontrol measures can negate the need for post-synthesis purification.This technique is represented schematically in FIG. 19, where a 3′exonuclease is introduced after a wash step to remove excess nucleotideanalogs and prior to linker cleavage. Such a cleaning step, as shown inFIG. 19, will reduce the number of oligonucleotides that are of anundesired length and/or sequence.

Another embodiment for using non-template dependentpolymerase/transferase enzymes would be to using protein engineering orprotein evolution to modify the enzyme to remain tightly bound andinactive to the nascent strand after each single nucleotideincorporation, thus preventing any subsequent incorporation until suchtime as the polymerase/transferase is released from the strand by use ofa releasing reagent/condition. Such modifications would be selected toallow the use of natural unmodified dNTPs instead of reversibleterminator dNTPs. Releasing reagents could be high salt buffers,denaturants, etc. Releasing conditions could be high temperature,agitation, etc. For instance, mutations to the Loop1 and SD1 regions ofTdT have been shown to dramatically alter the activity from atemplate-independent activity to more of a template dependent activity.Specific mutations of interest include but are not limited toΔ₃384/391/392, del loop1 (386→398), L398A, D339A, F401A, andQ402K403C404→E402R403S404. Other means of accomplishing the goal of apost-incorporation tight binding (i.e., single turnover) TdT enzymecould include mutations to the residues responsible for binding thethree phosphates of the initiator strand including but not limited toK261, R432, and R454.

Another embodiment for using non-template dependentpolymerase/transferase enzymes would be to use protein engineering orprotein evolution to modify the enzyme to accept 3-blocked reversibleterminators with high efficiency. Most naturally occurringpolymerase/transferase enzymes will not incorporate 3′-blockedreversible terminators due to steric constraints in the active site ofthe enzyme. Modifying either single or several aa residues in the activesite of the enzyme can allow the highly efficient incorporation of3′-blocked reversible terminators into a support bound initiator in aprocess completely analogous to that described above. Afterincorporation, the 3′-reversible terminator is removed with a deblockingreagent/condition thus generating a completely natural (scarless) singlestrand molecule ready for subsequent controlled extension reactions.There are residues close to the 3′-OH of the incoming dNTP whichexplains the propensity of TdT for incorporating ribonucleotidetriphosphates as readily as deoxyribonucleotide triphosphates; residuesincluding but not limited to those between β1 and β2 especially R334,Loop1, and those between α13 and α14, especially R454, are likelytargets for mutagenesis to accommodate the bulk of 3′-reversibleterminator groups and allow their efficient incorporation. In certainembodiments additional amino acid changes may be required to compensatefor these residue alterations made to accommodate a 3′-reversibleterminator. Another embodiment for using template-dependent polymeraseswould be to use the either 3′blocked or 3′unblocked dNTP analogs with aplurality of primer-template pairs attached to a solid support where thetemplate is a nucleic acid analog that supports polymerase mediatedprimer extension of any of the four bases as specified by the user.

Another embodiment for using non-template dependentpolymerase/transferase enzymes can use protein engineering or proteinevolution to modify the enzyme to optimize the use of each of the fourdifferent nucleotides or even different modified nucleotide analogs inan analog specific manner. Nucleotide specific or nucleotide analogspecific enzyme variants could be engineered to possess desirablebiochemical attributes like reduced K_(m) or enhanced addition ratewhich would further reduce the cost of the synthesis of desiredpolynucleotides.

Solid State Synthesis

The methods of the invention can be practiced under a variety ofreaction conditions, however the orderly construction and recovery ofdesired polynucleotides will, in most cases, require a solid support towhich the polynucleotides can be grown. In some embodiments, the methodsinclude the enzymatically-mediated synthesis of polynucleotides on asolid support. When used in conjunction with the NTP, linker, andinhibitor analogs discussed above, it is possible to construct specificpolynucleotide sequences of DNA as well as RNA by using, for example,TdT or poly(A) polymerase in an aqueous environment. As shown in FIG.13, the TdT can be used to effect the stepwise construction of custompolynucleotides by extending the polynucleotide sequence a stepwisefashion. As discussed previously, the inhibitor group of each NTP analogcauses the enzyme to stop with the addition of a nucleotide. After eachnucleotide extension step, the reactants are washed away from the solidsupport prior to the removal of the inhibitor by cleaving the linker,and then new reactants are added, allowing the cycle to start anew.

In certain embodiments, an additional quality control step may beincorporated in which the oligonucleotide or polynucleotide sequence isexposed to 3′ exonuclease after the nucleotidyl transferase mediatednucleotide analog extension step and before inhibitor cleavage. 3′exonuclease may degrade oligonucleotide or polynucleotide strands withan unblocked 3′ OH. In certain aspects, an uncleaved inhibitor (e.g., asteric inhibitor) may physically block the 3′ exonuclease from degradinga strand to which an uncleaved nucleotide analog has been successfullyincorporated. Such a quality control step may degrade only theoligonucleotides or polynucleotides which have unsuccessfullyincorporated the desired nucleotide analog, thereby eliminating anyerrors in the finished synthesized sequence potentially removing theneed for post-synthesis sequence verification. After 3′ exonucleaseexposure, the enzyme may be washed away before carrying on with theinhibitor cleavage step.

The 3′ exonuclease may act by shortening or completely degrading strandsthat have not successfully added the desired nucleotide analog. Strandsthat fail to be enzymatically extended at a given cycle will not have aterminal macromolecule-dNMP conjugate prior to the linker cleavage step.If a 3′-exonuclease is introduced at this stage, the full length strandcould be protected from degradation while “failure” strands will beshorted in length or potentially degraded completely to mononucleotidephosphates. The yield of long (>500 bases) synthetic DNA is dependent onhighly efficient reactions occurring at each and every cycle; both theenzymatic extension and the deblocking/self-elimination steps must occurat near quantitative yields. The introduction of a 3′-exonuclease afterthe enzymatic extension step but before the macromolecule terminatorcleavage step would have a positive impact on the nascent strand purityif the extension efficiency is less than quantitative (i.e., there arestrands that are not extended and therefore possess a natural unmodifiedterminal nucleotide).

*Conversely, a 3′-exonuclease step would have no impact on the qualityof the synthesis if the deblocking/elimination step is less thanquantitative because those strands would still be protected by themacromolecule terminator and fail to extend during the next extensionstep. Thus the actual improvement of the quality of the synthesis withthe addition of a 3′-exonuclease step can only be experimentallydetermined and then an assessment made if it is worth the additionalcost and cycle time. Since much of the cost of long ODNs made by theexisting phosphoramidite method is related to post-synthesis

At the conclusion of n cycles of extension-remove-deblocking-wash, thefinished full-length, single-strand polynucleotide is complete and canbe cleaved from the solid support and recovered for subsequent use inapplications such as DNA sequencing or PCR. Alternatively, the finished,full-length, single-strand polynucleotide can remain attached to thesolid support for subsequent use in applications such as hybridizationanalysis, protein or DNA affinity capture. In other embodiments,partially double-stranded DNA can be used as an initiator, resulting inthe synthesis of double-stranded polynucleotides.

Solid supports suitable for use with the methods of the invention mayinclude glass and silica supports, including beads, slides, pegs, orwells. In some embodiments, the support may be tethered to anotherstructure, such as a polymer well plate or pipette tip. In someembodiments, the solid support may have additional magnetic properties,thus allowing the support to be manipulated or removed from a locationusing magnets. In other embodiments, the solid support may be a silicacoated polymer, thereby allowing the formation of a variety ofstructural shapes that lend themselves to automated processing.

Synthesizers

To capitalize on the efficiency of the disclosed methods, an aqueousphase DNA synthesizer can be constructed to produce desiredpolynucleotides in substantial quantities. In one embodiment, asynthesizer will include four wells of the described NTP analogreagents, i.e., dCTP, dATP, dGTP, and dTTP, as well as TdT atconcentrations sufficient to effect polynucleotide growth. A pluralityof initiating sequences can be attached to a solid support that isdesigned to be repeatedly dipped into each of the four wells, e.g.,using a laboratory robot. The robot could be additionally programmed torinse the solid support in wash buffer between nucleotide additions,cleave the linking group by exposing the support to a deblocking agent,and wash the solid support a second time prior to moving the solidsupport to the well of the next desired nucleotide. With simpleprogramming, it is possible to create useful amounts of desirednucleotide sequences in a matter of hours, and with substantialreductions hazardous waste. Ongoing synthesis under carefully controlledconditions will allow the synthesis of polynucleotides with lengths inthe thousands of base pairs. Upon completion, the extension products arereleased from the solid support, whereupon they can be used as finishednucleotide sequences.

A highly parallel embodiment could consist of a series ofinitiator-solid supports on pegs in either 96 or 384 well formats thatcould be individually retracted or lowered so that the pegs can beindexed to contact the liquids in the wells in a controlled fashion. Thesynthesizer could thus consist of the randomly addressable peg device,four enzyme-dNTP analog reservoirs in the same format as the peg device(96 or 384 spacing), additional reagent reservoirs (washing, deblocking,etc.) in the same format as the peg device (96 or 384 spacing), and atransport mechanism (e.g., a laboratory robot) for moving the peg devicefrom one reservoir to another in a user programmable controlled butrandom access fashion. Care must be taken to avoid contaminating each ofthe four enzyme-dNTP reservoirs since the contents are reused throughoutthe entire synthesis process to reduce the cost of each polynucleotidesynthesis.

In alternative embodiments, the reagents (e.g., nucleotide analogs,enzymes, buffers) will be moved between solid supports, allowing thereagents to be recycled. For example a system of reservoirs and pumpscan move four different nucleotide analog solutions, wash buffers,and/or reducing agent solutions between one or more reactors in whichthe oligonucleotides will be formed. The reactors and pumps can beconventional, or the devices may be constructed using microfluidics.Because of the non-anhydrous (aqueous) nature of the process, no specialcare needs to be taken in the design of the hardware used to eliminateexposure to water. The synthesis process can take place with onlyprecautions to control evaporative loss. A highly parallel embodimentcould consist of a monolithic series of initiator-solid supports on pegsin either 96 or 384 well format that can be interfaced to a series ofwells in the same matching format. Each well would actually be areaction chamber that is fed by four enzyme-dNTP analog reservoirs andadditional reagent reservoirs (washing, deblocking, etc.) withappropriate valves. Provisions would be made in the fluidics logic torecover the enzyme-dNTP reactants in a pristine fashion after eachextension reaction since they are reused throughout the entire synthesisprocess to reduce the cost of each polynucleotide synthesis. In otherembodiments, a system of pipetting tips could be used to add and removereagents.

In certain aspects, polynucleotides may be synthesized usingmicrofluidic devices and/or inkjet printing technology. An exemplarymicrofluidic polynucleotide synthesis device is shown in FIG. 17 forillustrative purposes and not to scale. Microfluidic channels 255,including regulators 257, couple reservoirs 253 to a reaction chamber251 and an outlet channel 259, including a regulator 257 can evacuatewaste from the reaction chamber 251. Microfluidic devices forpolynucleotide synthesis may include, for example, channels 255,reservoirs 253, and/or regulators 257. Polynucleotide synthesis mayoccur in a microfluidic reaction chamber 251 which may include a numberof anchored synthesized nucleotide initiators which may include beads orother substrates anchored or bound to an interior surface of thereaction chamber and capable of releasably bonding a NTP analog orpolynucleotide initiator. The reaction chamber 251 may include at leastone intake and one outlet channel 259 so that reagents may be added andremoved to the reaction chamber 251. The microfluidic device may includea reservoir 253 for each respective NTP analog. Each of these NTP analogreservoirs 253 may also include an appropriate amount of TdT or anyother enzyme which elongates DNA or RNA strands without templatedirection. Additional reservoirs 253 may contain reagents forlinker/inhibitor cleavage and washing. These reservoirs 253 can becoupled to the reaction chamber 251 via separate channels 255 andreagent flow through each channel 255 into the reaction chamber 251 maybe individually regulated through the use of gates, valves, pressureregulators, or other means. Flow out of the reaction chamber 251,through the outlet channel 259, may be similarly regulated.

In certain instances, reagents may be recycled, particularly the NTPanalog-enzyme reagents. Reagents may be drawn back into their respectivereservoirs 253 from the reaction chamber 251 via the same channels 255through which they entered by inducing reverse flow using gates, valves,pressure regulators or other means. Alternatively, reagents may bereturned from the reaction chamber 251 to their respective reservoirs253 via independent return channels. The microfluidic device may includea controller capable of operating the gates, valves, pressure, or otherregulators 257 described above.

An exemplary microfluidic polynucleotide synthesis reaction may includeflowing a desired enzyme-NTP analog reagent into the reaction chamber251; after a set amount of time, removing the enzyme-NTP analog reagentfrom the reaction chamber 251 via an outlet channel 259 or a returnchannel; flowing a wash reagent into the reaction chamber 251; removingthe wash reagent from the reaction chamber 251 through an outlet channel259; flowing a de-blocking or cleavage reagent into the reaction chamber251; removing the de-blocking or cleavage reagent from the reactionchamber 251 via an outlet channel 259 or a return channel; flowing awash reagent into the reaction chamber 251; removing the wash reagentfrom the reaction chamber 251 through an outlet channel 259; flowing theenzyme-NTP analog reagent including the next NTP in the desired sequenceto be synthesized into the reaction chamber 251; and repeating until thedesired polynucleotide has been synthesized. After the desiredpolynucleotide has been synthesized, it may be released from thereaction chamber anchor or substrate and collected via an outlet channel259 or other means.

In certain aspects, reagents and compounds, including NTP analogs, TdTand/or other enzymes, and reagents for linker/inhibitor cleavage and/orwashing may be deposited into a reaction chamber using inkjet printingtechnology or piezoelectric drop-on-demand (DOD) inkjet printingtechnology. Inkjet printing technology can be used to form droplets,which can be deposited, through the air, into a reaction chamber.Reagent droplets may have volumes in the picoliter to nanoliter scale.Droplets may be introduced using inkjet printing technology at a varietyof frequencies including 1 Hz, 10, Hz, 100 Hz, 1 kHz, 2 kHz, and 2.5kHz. Various reagents may be stored in separate reservoirs within theinkjet printing device and the inkjet printing device may deliverdroplets of various reagents to various discrete locations including,for example, different reaction chambers or wells within a chip. Incertain embodiments, inkjet and microfluidic technologies may becombined wherein certain reagents and compounds are delivered to thereaction chamber via inkjet printing technology while others aredelivered via microfluidic channels or tubes. An inkjet printing devicemay be controlled by a computing device comprising at least anon-transitory, tangible memory coupled to a processor. The computingdevice may be operable to receive input from an input device including,for example, a touch screen, mouse, or keyboard and to control when andwhere the inkjet printing device deposits a droplet of reagent, thereagent it deposits, and/or the amount of reagent deposited.

In certain instances, a desired polynucleotide sequence may be enteredinto the computing device through an input device wherein the computingdevice is operable to perform the necessary reactions to produce thedesired polynucleotide sequence by sequentially depositing theappropriate NTP analog, enzyme, cleavage reagent, and washing reagent,in the appropriate order as described above.

After synthesis, the released extension products can to be analyzed byhigh resolution PAGE to determine if the initiators have been extendedby the anticipated number of bases compared to controls. A portion ofthe recovered synthetic DNA may also be sequenced to determine if thesynthesized polynucleotides are of the anticipated sequence.

Because the synthesizers are relatively simple and do not require thetoxic components needed for phosphoramidite synthesis, synthesizers ofthe invention will be widely accessible for research institutions,biotechs, and hospitals. Additionally, the ability to reuse/recyclereagents will reduce the waste produced and help reduce the costs ofconsumables. The inventors anticipate that the methods and systems willbe useful in a number of applications, such as DNA sequencing, PCR, andsynthetic biology.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

The invention claimed is:
 1. A method for synthesizing anoligonucleotide, comprising: exposing an oligonucleotide attached to asolid support to a nucleotide analog in the presence of a nucleotidyltransferase enzyme and in the absence of a nucleic acid template suchthat the nucleotide analog is incorporated into the oligonucleotide,wherein the nucleotide analog comprises a nucleotide coupled, by acleavable linker, to an inhibitor that sterically prevents thenucleotidyl transferase from catalyzing incorporation of either anatural nucleotide or a nucleotide analog into said oligonucleotideuntil said inhibitor is removed by cleavage of said cleavable linker,and wherein the inhibitor is a macromolecule greater than 50 Å indiameter.
 2. The method of claim 1, wherein the nucleotide analogcomprises the following structure:


3. The method of claim 1, wherein the nucleotide analog comprises thefollowing structure:


4. A method for synthesizing an oligonucleotide, comprising: exposing anoligonucleotide attached to a solid support to a nucleotide analog inthe presence of a nucleotidyl transferase enzyme and in the absence of anucleic acid template such that the nucleotide analog is incorporatedinto the oligonucleotide, wherein the nucleotide analog comprises anucleotide coupled, by a cleavable linker, to an inhibitor thatsterically prevents the nucleotidyl transferase from catalyzingincorporation of a natural nucleotide or a nucleotide analog into saidoligonucleotide until said inhibitor is removed by cleavage of saidcleavable linker, and wherein the inhibitor comprises the followingstructure:


5. A method for synthesizing an oligonucleotide, comprising: exposing anoligonucleotide attached to a solid support to a nucleotide analog inthe presence of a nucleotidyl transferase enzyme and in the absence of anucleic acid template such that the nucleotide analog is incorporatedinto the oligonucleotide, wherein the nucleotide analog comprises anucleotide coupled, by a cleavable linker, to an inhibitor thatsterically prevents the nucleotidyl transferase from catalyzingincorporation of a natural nucleotide or a nucleotide analog into saidoligonucleotide until said inhibitor is removed by cleavage of saidcleavable linker, and wherein the inhibitor comprises a polypeptoid. 6.A method for synthesizing an oligonucleotide, comprising: exposing anoligonucleotide attached to a solid support to a nucleotide analog inthe presence of a nucleotidyl transferase enzyme and in the absence of anucleic acid template such that the nucleotide analog is incorporatedinto the oligonucleotide, wherein the nucleotide analog comprises anucleotide coupled, by a cleavable linker, to an inhibitor thatsterically prevents the nucleotidyl transferase from catalyzingincorporation of a natural nucleotide or a nucleotide analog into saidoligonucleotide until said inhibitor is removed by cleavage of saidcleavable linker, and wherein the inhibitor comprises a polymer.
 7. Themethod of claim 6, wherein the polymer comprises polyethylene glycol. 8.A method for synthesizing an oligonucleotide, comprising: exposing anoligonucleotide attached to a solid support to a nucleotide analog inthe presence of a nucleotidyl transferase enzyme and in the absence of anucleic acid template such that the nucleotide analog is incorporatedinto the oligonucleotide, wherein the nucleotide analog comprises anucleotide coupled, by a cleavable linker, to an inhibitor thatsterically prevents the nucleotidyl transferase from catalyzingincorporation of a natural nucleotide or a nucleotide analog into saidoligonucleotide until said inhibitor is removed by cleavage of saidcleavable linker, and wherein the inhibitor is a protein.
 9. A methodfor synthesizing an oligonucleotide, comprising: exposing anoligonucleotide attached to a solid support to a nucleotide analog inthe presence of a nucleotidyl transferase enzyme and in the absence of anucleic acid template such that the nucleotide analog is incorporatedinto the oligonucleotide, wherein the nucleotide analog comprises anucleotide coupled, by a cleavable linker, to an inhibitor thatsterically prevents the nucleotidyl transferase from catalyzingincorporation of a natural nucleotide or a nucleotide analog into saidoligonucleotide until said inhibitor is removed by cleavage of saidcleavable linker, and wherein the inhibitor is a nanoparticle.
 10. Themethod of claim 1, wherein the nucleic acid attached to the solidsupport is exposed to the nucleotide analog in the presence of anaqueous solution having a pH between about 6.5 and 8.5.
 11. The methodof claim 1, the nucleic acid attached to the solid support is exposed tothe nucleotide analog in the presence of an aqueous solution at atemperature between about 35 and 39° C.
 12. The method of claim 1,wherein the solid support is a bead, a well, or a peg.
 13. The method ofclaim 1, wherein the nucleic acid is single stranded.
 14. The method ofclaim 1, wherein the cleavable linker comprises a moiety that forms acyclic by-product when cleaved from the nucleotide analog.
 15. Themethod of claim 1, further comprising: cleaving the cleavable linker inorder to produce a native nucleotide; and exposing the native nucleotideto a second nucleotide analog in the presence of a nucleotidyltransferase enzyme and in the absence of a nucleic acid template. 16.The method of claim 1, further comprising exposing the oligonucleotideto an exonuclease enzyme.
 17. The method of claim 1, further comprisingproviding an aqueous solution comprising the nucleotide analog and thenucleotidyl transferase enzyme.
 18. The method of claim 1, wherein theinhibitor comprises a charged moiety.
 19. The method of claim 1, whereinthe nucleotide analog comprises a ribose sugar or a deoxyribose sugar.20. The method of claim 1, wherein the nucleotide substrate comprises abase selected from the group consisting of adenine, guanine, cytosine,thymine, and uracil.
 21. The method of claim 1, wherein the nucleotidyltransferase comprises a protein sequence that is at least about 90%identical to SEQ ID NO. 1, SEQ ID NO. 3, or SEQ ID NO.
 5. 22. The methodof claim 1, wherein the nucleotidyl transferase originates from anorganism having a nucleotide sequence that is at least about 90%identical to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO.
 6. 23. The methodof claim 1, wherein the nucleotide analog is delivered in a droplet.